Supporting Information Synthesis and anti-hiv profile of a novel tetrahydroindazolylbenzamide derivative obtained by oxazolone chemistry Angela Scala,,* Anna Piperno, Nicola Micale, Frauke Christ, Zeger Debyser Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale F. Stagno D Alcontres 31, Messina I-98166, Italy Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, UZ St. Rafael, Kapucijnenvoer 33, BE-3000 Leuven, Belgium Table of content: - Materials and Methods 2 - Synthetic procedures...2 - Drug susceptibility assay..3 - Quantification of different HIV-1 DNA species during HIV infection by real-time PCR..3 - References.3-1 H and 13 C NMR spectra 4 1
Materials and Methods All reagents and solvents were obtained from commercial suppliers and purified before use if necessary. 1 H and 13 C-NMR spectra were obtained on a Varian 500 MHz spectrometer. The chemical shifts (δ) and coupling constants (J) are expressed in ppm and hertz, respectively. Merck Kieselgel 60F254 plates were used for TLC. Compounds 1 and 3 were prepared according to literature. 1-2 Synthetic procedures Synthesis of N-(1-(1-(4-cyanophenyl)-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-3- yl)ethyl)benzamide (5): To a stirred solution of 3 (500 mg, 1.6 mmol) in abs EtOH (30 ml), hydrazine 4 (2 mmol) was added. The mixture was heated at reflux under stirring overnight. The progress of the reaction was monitored by TLC (CH 2 Cl 2 /AcOEt 9:1). After completion of reaction, the mixture was evaporated under vacuum affording 5 as yellowish oil in a nearly quantitative yield. 1 H NMR (500 MHz, CDCl 3 ): δ = 1.10 (s, 3H, CH 3 ), 1.18 (s, 3H, CH 3 ), 1.55 (d, J= 7.1 Hz, 3H, CH 3 ), 2.57, 2.48 (AB system, J H,H = 16.3 Hz, 2H, CH 2 ), 2.92, 2.83 (AB system, J H,H = 16.3 Hz, 2H, CH 2 ), 5.77 (dq, J= 7.1 Hz, 9.2 Hz, 1H, CH), 7.42-7.50 (m, 3H Ar), 7.69 (d, J= 8.5 Hz, 2H Ar), 7.82 (d, J= 8.5 Hz, 2H Ar), 7.89 (d, J= 7.1 Hz, 2H Ar), 8.74 (d, NH, J= 9.2 Hz); 13 C NMR (125 MHz, CDCl 3 ): δ = 21.9, 27.8, 28.7, 35.9, 37.5, 43.8, 52.0, 111.7, 116.7, 117.8, 123.8, 127.1, 128.5, 131.4, 133.4, 134.4, 141.6, 150.7, 155.6, 166.2, 194.4. HRMS-EI (m/z) [M] + calcd for C 25 H 24 N 4 O 2 412.1899, found 412.1897. Synthesis of N-(1-(1-(4-carbamoylphenyl)-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-3- yl)ethyl)benzamide (6): Compound 5 (250 mg, 0.6 mmol) was dissolved in 3 ml of absolute ethanol. DMSO (1 ml) was added under stirring followed by 2 ml of NaOH solution 1M at 10 C within 10 min. After 10 min, 1.5 ml of H 2 O 2 50 wt% was added dropwise over an extended time at 10 C to control the exotherm reaction (Caution: hydrogen peroxide can be hazardous. Use appropriate precautions!). The mixture was stirred at room temperature for 24 h. The reaction was cooled at 5 C and, after 30 min 5 ml of a cold solution of Na 2 S 2 O 3 al 10% and 4 ml of distilled water were added. The solid residue was filtered and washed with water and ethanol to give 6 as orange-yellow solid; yield: 92 %; 1 H NMR (500 MHz, DMSO-d 6 ): δ: 1.03 (s, 3H, CH 3 ), 1.04 (s, 3H, CH 3 ), 1.47 (d, J= 7.1 Hz, 3H, CH 3 ), 2.44 (s, 2H, CH 2 ), 2.97 (s, 2H, CH 2 ), 5.55 (dq, J= 7.1 Hz, 8.8 Hz, 1H, CH), 7.46-7.55 (m, 3H Ar), 7.68 (d, J= 8.4 Hz, 2H Ar), 7.83 (d, J= 8.4 Hz, 2H Ar), 8.03 (d, J= 8.4 Hz, 2H Ar), 8.08 (s br, NH 2 ), 8.88 (d, NH, J= 8.8 Hz); 13 C NMR (125 MHz, DMSO-d 6 ): δ = 21.5, 28.0, 28.1, 35.8, 36.6, 43.8, 52.1, 110.0, 115.7, 123.4, 127.5, 128.8, 129.2, 131.7, 134.0, 134.8, 140.5, 151.4, 165.6, 167.3, 194.0. HRMS-EI (m/z) [M] + calcd for C 25 H 26 N 4 O 3 430.2005, found 430.2007. Synthesis of 4-(3-(1-aminoethyl)-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzoic acid (7): Compound 5 (300 g, 0.7 mmol) was dispersed in a mixture of 6N HCl (40 ml) and glacial acetic acid (40 2
ml). The mixture was heated at reflux for 2 days. After cooling, the aqueous solution was extracted with ethyl acetate (3 50 ml). The combined organic layers were dried over anhydrous Na 2 SO 4, filtered, evaporated under reduced pressure and the residue was treated with acetone to remove benzoic acid. The filtrate was evaporated under vacuum affording compound 7 as a yellowish solid; yield: 80 %; 1 H NMR (500 MHz, CD 3 OD): δ = 1.11 (s, 3H, CH 3 ), 1.12 (s, 3H, CH 3 ), 1.70 (d, J= 6.6 Hz, 3H, CH 3 ), 2.52 (s, 2H, CH 2 ), 3.03 (s, 2H, CH 2 ), 4.90 (m, 1H, CH), 7.76 (d, J= 8.4 Hz, 2H Ar), 8.21 (t, J= 8.4, 2H Ar); 13 C NMR (125 MHz, CD 3 OD): δ = 17.2, 26.8, 26.9, 35.6, 36.3, 45.2, 51.2, 115.7, 123.2, 130.6, 130.7, 141.5, 149.9, 151.9, 167.1, 195.0. HRMS-EI (m/z) [M] + calcd for C 18 H 21 N 3 O 3 327.1583, found 327.1585. Drug susceptibility assay. The inhibitory effect of antiviral drugs on the HIV-induced CPE in MT-4 cell culture was determined by the MTT-assay. 3 This assay is based on the reduction of the yellow colored 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase of metabolically active cells to a blue formazan derivative, which can be measured spectrophotometrically. The 50% cell culture infective dose of the HIV strains was determined by titration of the virus stock using MT-4 cells. For the drug susceptibility assays, MT-4 cells were infected with 100 to 300 x 50% cell culture infective doses of the HIV strains in the presence of fivefold serial dilutions of the antiviral drugs. The concentration of the compound achieving 50% protection against the CPE of HIV, which is defined as the 50% effective concentration (IC 50 ), was determined. The concentration of the compound destroying 50% of the MT-4 cells, which is defined as the 50% cytotoxic concentration (CC 50 ), was determined as well. Quantification of different HIV-1 DNA species during HIV infection by real-time PCR. HeLaP4 cells (1.0 x 10 6 cells per well in a 6-well plate) were incubated with HIV-1 NL4.3 (corresponding to 1 µg of p24) in the absence or presence of the test compounds. Inhibitors were added to the cells during infection. After 2 h incubation at 37 C the cells were washed 3 times with phosphate buffered saline. When infection medium was replaced with new medium, fresh inhibitors were added. In each 6-well plate, uninfected HeLaP4 cells were incubated in parallel. Each time a sample was prepared for quantitative PCR (Q-PCR) analysis, an aliquot of uninfected cells was prepared as well. DNA extractions and quantification of late reverse transcripts, two-long terminal repeat (2-LTR) circles, and integrant were done as described earlier. 4 2-LTR forward and reverse primers though were replaced by the following primers: LTR forward 5 - AACTAGGGAACCCACTGCTTAAG-3 and LTR reverse 5 -TCCACAGATCAAGGATCTCTTGTC-3. References 1. Piperno, A.; Scala, A.; Risitano F.; Grassi, G. Oxazol-5-(4H)-Ones. Part 1. Synthesis and Reactivity as 1,3-dipoles. Curr. Org. Chem., (2014), 18, 2691 2710. 2. Cordaro, M.; Grassi, G.; Risitano, F.; Scala, A. A new construction of diversely functionalized oxazoles from enolizable cyclic 1,3-dicarbonyls and 5(4H)-oxazolones. Synlett (2009), 1:103 105. 3. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.; Desmyter, J.; De Clercq, E. Rapid and auto-mated tetrazolium-based colorimetric assay for the detection of anti-hiv compounds. J Virol Methods (1988) 20:309-321. 4. B. Van Maele, J. De Rijck, E. De Clercq, Z. Debyser. Impact of the Central Polypurine Tract on the Kinetics of Human Immunodeficiency Virus Type 1 Vector Transduction. J Virol. (2003), 77, 4685-4694. 3
1 H and 13 C NMR spectra Compound 5 1 H NMR (500 MHz, CDCl 3 ): Compound 5 13 C NMR (125 MHz, CDCl 3 ): 4
Compound 6 1 H NMR (500 MHz, DMSO-d 6 ): Compound 6 13 C NMR (125 MHz, DMSO-d 6 ): 5
Compound 7 1 H NMR (500 MHz, CD 3 OD): Compound 7 13 C NMR (125 MHz, CD 3 OD): 6